Received on January 18, 2008
Accepted on January 28, 2008

Automation and Analytical Techniques

Automation and Analytical Techniques: Measurement of Folate in Fresh and Archival Serum Samples as p-Aminobenzoylglutamate Equivalents

¹ LOCUS for Homocysteine and Related Vitamins, and Section for Pharmacology, Institute of Medicine, University of Bergen, Norway
² LOCUS for Homocysteine and Related Vitamins, and Section for Pharmacology, Institute of Medicine, University of Bergen, and Haukeland University Hospital, Bergen, Norway

^* To whom correspondence should be addressed. E-mail: rita.hannisdal{at}farm.uib.no.

BACKGROUND: The development of accurate and precise folate assays has been difficult, mainly because of folate instability. Large interassay and interlaboratory differences have been reported. We therefore developed a serum folate assay that measures folate and putative degradation products as p-aminobenzoylglutamate (pABG) equivalents following oxidation and acid hydrolysis.

METHODS: Serum was deproteinized with acid in the presence of 2 internal calibrators ([¹³C₂]pABG and [¹³C₅]5-methyltetrahydrofolate). 5-Methyltetrahydrofolate and other folate species in serum were converted to pABG by oxidation and mild acid hydrolysis. pABG and its internal calibrators were quantified by tandem liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS).

RESULTS: The limit of quantification was 0.25 nmol/L, and the assay was linear in the range 0.25–96 nmol/L, which includes the 99.75 percentile for serum folate concentrations in healthy blood donors. Within- and between-day imprecision was 5%. We detected no residual folate in serum samples after sample preparation. Folate concentrations in fresh serum samples obtained with the pABG assay and with a microbiologic assay showed good agreement (r = 0.96). In stored samples containing low folate concentrations due to folate degradation, the pABG assay yielded substantially higher folate concentrations than the microbiologic assay.

CONCLUSIONS: The pABG assay combines automated sample preparation with LC-MS/MS analysis. It allows measurement of folate not only in fresh samples of serum/plasma but also in stored samples in which the folate has become oxidized and degraded to an extent that it cannot be assayed with traditional folate assays.